Acyclic linker-containing oligonucleotides and uses thereof
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example 1
Synthesis of Acyclic Precursors Suitable for their Incorporation into Oligonucleotides
Precursor to Acyclic Residue IIb
[0125] Dimethoxytrityl-O—CH2CH2CH2CH2—O—P(Ni—Pr2)OCH2CH2CN (1) was purchased from ChemGenes Corp. (Ashland, Mass.), and was used as received for the synthesis of AON-X-AON chimeras (See Example 3).
Precursor to Acyclic Residues IIc and IId
[0126] The acyclic nucleoside residues (herein referred to as 2′,3′-seconucleotides) consist of a 1-[1,5-dihydroxy-4(S)-hydroxymethyl-3-oxapent-2(R)-yl]-uracil unit which has been appropriately protected and functionalized for oligonucleotide incorporation as described below.
Step A. Synthesis of 5′-monomethoxytrityl-2′,3′-seco-β-D-ribouracil (2)
[0127]
[0128] To a 0.1 M solution of 5′-monomethoxytrityluridine (5′-MMT-rU, 5.16 g, 10 mmol; prepared as described in T. Wu, K. K. Ogilvie, R. T. Pon. 1989. “Prevention of Chain Cleavage in the Chemical Synthesis of 2′-Silylated Oligoribonucleotides.”Nucleic Acids Res., 3501-17.) in d...
example 2
Preparation of AONs Constructed from 2′-deoxy-2′-fluoro-β-D-arabinonucleotides (FANA) Flanking an Acyclic Butanediol or Secouridine Residues
1. Synthesis of FANA-X-FANA Chimeras, where X=Butanediol Linker=IIb (Y=Z=Oxygen; n=4)
[0136] The synthesis of PDE-FANA oligomers was conducted as previously described (Damha et al. J. Am. Chem. Soc. 1998, 120, 12976; Wilds, C. J. & Damha, M. J. Nucleic Acids Res. 2000, 28, 3625). Their structure was confirmed via Maldi-TOF mass spectrometry.
[0137] Synthesis of PDE-(FANA-But-FANA) chimeras were performed on a 1 micromole scale using an Expedite 8909 DNA-synthesizer. Long-chain alkylamine controlled-pore glass (LCAA-CPG) was used as the solid support. The synthesis cycle consisted of the following steps: [0138] 1)-Detritylation of nucleoside / tide bound to CPG (3% trichloroacetic acid / dichloroethane): 150 sec for MMT; 60 sec for DMT removal. [0139] 2) Coupling of 2′-F-arabinonucleoside or dimethoxytrityl-butanediol phosphoramidite monomers: 15 ...
example 3
Expression and Purification of Human RNase HII
Expression of Human RNase HII
[0159] A hrnh gene fragment from pcDNA / GS / hrnh (Invitrogen) was obtained by PCR using the primers: AGC TAT CTC GAG ATG AGC TGG CTT CTG TTC CTG GCC (XhoI) (SEQ ID NO: 30), and GGC CGC AAG CTT TCA GTC TTC CGA TTG TTT AGC TCC (HindIII) (SEQ ID NO: 31). This fragment was cloned in the XhoI / HindIII sites of the bacterial expression vector pBAD / His (Invitrogen). Recombinant human RNase HII from pBAD / His / hrnh expression plasmid was purified as follows. To overcome codon bias in E. coli, we transformed the expression vector in E. coli BL21 codonplus (Stratagene) and cultured in LB broth containing 100 μg / ml ampicillin at 37° C. until the OD600 reached 0.5-0.6. Then, the recombinant protein was induced with 0.002% arabinose for 4 h.
Purification of Human RNase HII
[0160] After induction, E. coli cells were harvested by centrifugation, washed in chilled wash buffer (100 mM phosphate, pH 8.0, 300 mM NaCl), resuspen...
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